In the present study, oil palm mesocarp fibers (OPMF), an agroindustrial residue from the production of palm oil, were used to obtain cellulose nanowhiskers. They were obtained from bleaching of fibers, followed by hydrolysis using sulfuric acid and microfluidization, to control the length of cellulose nanowhiskers and avoid a decrease in thermal stability with extended acid hydrolysis time. The results showed that the nanowhiskers obtained by acid hydrolysis for 105 min resulted in structures with an average length (L) of 117 +/- 54 nm and diameter (D) of 10 +/- 5 nm. After 105 min of acid hydrolysis, the suspension was dialyzed and the neutral suspension was subjected to microfluidization. At this time the nanowhiskers presented the same dimensions, even with the fibrils disintegration of both amorphous and crystalline phases, during the microfluidization. However, after microfluidization, the sample presented a more stable suspension, but the crystallinity decreased. Increasing the hydrolysis time from 105 to 140 min, more sulfonated nanowhiskers were obtained, presenting lower thermal stability, but higher crystallinity than the microfluidized sample. Furthermore, this study proved that it is possible to obtain cellulose nanowhiskers from oil palm mesocarp fibers, an agroindustrial residue from the palm oil production, helping to reduce the environmental impact of this waste, as well as providing the obtaining of a high value-added product.